Figure 4

Figure 4

The effect of cold treatment on CRISPR/Cas9 mutagenesis efficiency in zebrafish embryos. Incubating zebrafish embryos in 12 °C immediately after Cas9/sgRNA injections increases the mutagenesis efficiency compared to embryos incubated in 28 °C. (a) Representative image of the heteroduplex motility assay (HMA) to analyze the mutagenesis efficiency of Cas9/sgRNA(ca6) injected embryos incubated in 12 °C versus 28 °C. The image shows the HMA result for one uninjected (control) embryo incubated in 28 °C, one uninjected embryo incubated in 12 °C, three Cas9/sgRNA(ca6) injected individuals incubated in 28 °C (Cas9 28 °C) and four Cas9/sgRNA(ca6) injected individuals incubated in 12 °C (Cas9 12 °C). (b) Mutant band intensities for control and Cas9/sgRNA(ca6) injected embryos. Figure represents combined results from 2 independent experiments, n(Cas9 12 °C) = 76, n(Cas9 28 °C) = 72, n(control 12 °C) = 54, n(control 28 °C) = 50. (c) Representative image of the heteroduplex motility assays (HMA) carried out to analyze the mutagenesis efficiency of Cas9/sgRNA(sema4gb) injected embryos incubated in 12 °C versus 28 °C. The image shows the HMA result for one uninjected (control) embryo incubated in 28 °C, one uninjected embryo incubated in 12 °C, three Cas9/sgRNA(sema4gb) injected individuals incubated in 28 °C (Cas9 28 °C) and four Cas9/sgRNA(sema4gb) injected individuals incubated in 12 °C (Cas9 12 °C). (d) Mutant band intensities for control and Cas9/sgRNA(sema4gb) injected embryos. Figure represents combined results from 2 independent experiments, n(Cas9/12 °C) = 68, n(Cas9/28 °C) = 66, n(control 12 °C) = 52, n(control 28 °C) = 48. (e) Number of mutant bands in 12 °C or 28 °C treated, Cas9/sgRNA(ca6) injected embryos, after heteroduplex formation, n(Cas9/12 °C) = 54, n(Cas9/28 °C) = 49. (f) Number of mutant bands in 12 °C or 28 °C treated, Cas9/sgRNA(sema4gb) injected embryos, after heteroduplex formation, n(Cas9/12 °C) = 52, n(Cas9/28 °C) = 47. Error bars represent SD, *p < 0.05, **p < 0.01, ***p < 0.001), WT Wild-type band.